Golden bananas in the field: elevated fruit pro‐vitamin A from the expression of a single banana transgene

Vitamin A deficiency remains one of the world's major public health problems despite food fortification and supplements strategies. Biofortification of staple crops with enhanced levels of pro‐vitamin A (PVA) offers a sustainable alternative strategy to both food fortification and supplementation. As a proof of concept, PVA‐biofortified transgenic Cavendish bananas were generated and field trialed in Australia with the aim of achieving a target level of 20 μg/g of dry weight (dw) β‐carotene equivalent (β‐CE) in the fruit. Expression of a Fe'i banana‐derived phytoene synthase 2a (MtPsy2a) gene resulted in the generation of lines with PVA levels exceeding the target level with one line reaching 55 μg/g dw β‐CE . Expression of the maize phytoene synthase 1 (ZmPsy1) gene, used to develop ‘Golden Rice 2’, also resulted in increased fruit PVA levels although many lines displayed undesirable phenotypes. Constitutive expression of either transgene with the maize polyubiquitin promoter increased PVA accumulation from the earliest stage of fruit development. In contrast, PVA accumulation was restricted to the late stages of fruit development when either the banana 1‐aminocyclopropane‐1‐carboxylate oxidase or the expansin 1 promoters were used to drive the same transgenes. Wild‐type plants with the longest fruit development time had also the highest fruit PVA concentrations. The results from this study suggest that early activation of the rate‐limiting enzyme in the carotenoid biosynthetic pathway and extended fruit maturation time are essential factors to achieve optimal PVA concentrations in banana fruit.     

Beyond host-pathogen interactions: microbial defense strategy in the host environment

Many extracellular pathogenic bacteria colonize human or animal bodies through evasion of the host immune system, a process called host-pathogen interaction. What happens when other intruders try to invade the same host and try to establish themselves in the same niche is largely unknown. In one well-studied case, Pseudomonas aeruginosa is known to secrete the protein azurin as a weapon against such invaders as cancers, parasites and viruses. The production of such weapons by pathogenic bacteria could provide important insights into how a pathogen responds in the post-colonization state to impede other intruders for its own survival. Moreover, these molecules might find use in the pharmaceutical industry as next-generation therapeutics.     

Effects of arbuscular mycorrhizal fungi on seedling growth and development of two wetland plants, Bidens frondosa L., and Eclipta prostrata (L.) L., grown under three levels of water availability

To identify the importance of arbuscular mycorrhizal fungi (AMF) colonizing wetland seedlings following flooding, we assessed the effects of AMF on seedling establishment of two pioneer species, Bidens frondosa and Eclipta prostrata grown under three levels of water availability and ask: (1) Do inoculated seedlings differ in growth and development from non-inoculated plants? (2) Are the effects of inoculation and degree of colonization dependent on water availability? (3) Do plant responses to inoculation differ between two closely related species? Inoculation had no detectable effects on shoot height, or plant biomass but did affect biomass partitioning and root morphology in a species-specific manner. Shoot/root ratios were significantly lower in non-inoculated E. prostrata plants compared with inoculated plants (0.381 ± 0.066 vs. 0.683 ± 0.132). Root length and surface area were greater in non-inoculated E. prostrata (259.55 ± 33.78 cm vs. 194.64 ± 27.45 cm and 54.91 ± 7.628 cm² vs. 46.26 ± 6.8 cm², respectively). Inoculation had no detectable effect on B. frondosa root length, volume, or surface area. AMF associations formed at all levels of water availability. Hyphal, arbuscular, and vesicular colonization levels were greater in dry compared with intermediate and flooded treatments. Measures of mycorrhizal responsiveness were significantly depressed in E. prostrata compared with B. frondosa for total fresh weight (−0.3 ± 0.18 g vs. 0.06 ± 0.06 g), root length (−0.78 ± 0.28 cm vs.−0.11 ± 0.07 cm), root volume (−0.49 ± 0.22 cm³ vs. 0.06 ± 0.07 cm³), and surface area (−0.59 ± 0.23 cm² vs.−0.03 ± 0.08 cm²). Given the disparity in species response to AMF inoculation, events that alter AMF prevalence in wetlands could significantly alter plant community structure by directly affecting seedling growth and development.     

The yeast Apq12 protein affects nucleocytoplasmic mRNA transport

An important step in mRNA biogenesis is the export of mRNA from the nucleus to the cytoplasm. In this work, we provide evidence that the previously uncharacterized gene APQ12 functions in nucleocytoplasmic mRNA transport in Saccharomyces cerevisiae. First, apq12delta strains manifest 3' hyperadenylated mRNA similar to other previously characterized RNA export mutants. Second, bulk poly(A)+ RNA is retained in the nucleus in apq12delta cells. Third, an Apq12p-GFP chimeric protein is localized to the nuclear periphery. Fourth, mRNA in apq12delta cells is stabilized, consistent with a defect in the rate of nuclear export. Interestingly, apq12delta mutants are severely compromised for growth and display atypical cell morphology. Because this aberrant cell morphology is not seen with other viable export mutants, Apq12p must have either an additional cellular function, or preferentially impinge on the export of mRNAs regulating cell growth. Together, these findings support a role for APQ12 in nucleocytoplasmic transport of mRNA.     

Salvaging Pyrococcus furiosus Protein Targets at SECSG

Proteins derived from the coding regions of Pyrococcus furiosus are targets for three-dimensional X-ray and NMR structure determination by the Southeast Collaboratory for Structural Genomics (SECSG). Of the 2200 open reading frames (ORFs) in this organism, 220 protein targets were cloned and expressed in a high-throughput (HT) recombinant system for crystallographic studies. However, only 96 of the expressed proteins could be crystallized and, of these, only 15 have led to structures. To address this issue, SECSG has recently developed a two-tier approach to protein production and crystallization. In this approach, tier-1 efforts are focused on producing protein for new Pfu(italics?) targets using a high-throughput approach. Tier-2 protein production efforts support tier-1 activities by (1) producing additional protein for further crystallization trials, (2) producing modified protein (further purification, methylation, tag removal, selenium labeling, etc) as required and (3) serving as a salvaging pathway for failed tier-1 proteins. In a recent study using this two-tiered approach, nine structures were determined from a set of 50 Pfu proteins, which failed to produce crystals suitable for X-ray diffraction analysis. These results validate this approach and suggest that it has application to other HT crystal structure determination applications.     

Molecular mapping of QTLs for resistance to Gibberella ear rot, in corn, caused by Fusarium graminearum.

Gibberella ear rot, caused by the fungus Fusarium graminearum Schwabe, is a serious disease of corn (Zea mays) grown in northern climates. Infected corn is lower yielding and contains toxins that are dangerous to livestock and humans. Resistance to ear rot in corn is quantitative, specific to the mode of fungal entry (silk channels or kernel wounds), and highly influenced by the environment. Evaluations of ear rot resistance are complex and subjective; and they need to be repeated over several years. All of these factors have hampered attempts to develop F. graminearum resistant corn varieties. The aim of this study was to identify molecular markers linked to the genes for resistance to Gibberella ear rot. A recombinant inbred (RI) population, produced from a cross between a Gibberella ear rot resistant line (CO387) and a susceptible line (CG62), was field-inoculated and scored for Gibberella ear rot symptoms in the F4, F6, and F7 generations. The distributions of disease scores were continuous, indicating that resistance is probably conditioned by multiple loci. A molecular linkage map, based on segregation in the F5 RI population, contained 162 markers distributed over 10 linkage groups and had a total length of 2237 cM with an average distance between markers of 13.8 cM. Composite interval mapping identified 11 quantitative trait loci (QTLs) for Gibberella ear rot resistance following silk inoculation and 18 QTLs following kernel inoculation in 4 environments that accounted for 6.7%-35% of the total phenotypic variation. Only 2 QTLs (on linkage group 7) were detected in more than 1 test for silk resistance, and only 1 QTL (on linkage group 5) was detected in more than 1 test for kernel resistance, confirming the strong influence of the environment on these traits. The majority of the favorable alleles were derived from the resistant parent (CO387). The germplasm and markers for QTLs with significant phenotypic effects may be useful for marker-assisted selection to incorporate Gibberella ear rot resistance into commercial corn cultivars.     

Use-dependent plasticity in barrel cortex: intrinsic signal imaging reveals functional expansion of spared whisker representation into adjacent deprived columns

We used optical imaging of intrinsic cortical signals, elicited by whisker stimulation, to define areas of activation in primary sensory cortex of normal hamsters and hamsters subjected to neonatal follicle ablation at postnatal day seven (P7). Follicle ablations were unilateral, and spared either C-row whiskers or the second whisker arc. This study was done to determine if the intrinsic cortical connectivity pattern of the barrel cortex, established during the critical period, affects the process of representational plasticity that follows whisker follicle ablation. Additionally, we tested the ability to monitor such changes in individual cortical whisker representations using intrinsic signal imaging. Stimulation of a single whisker yielded peak activation of a barrel-sized patch in the somatotopically appropriate location in normal cortex. In both row and arc-spared animals, functional representations corresponding to spared follicles were significantly stronger and more oblong than normal. The pattern of activation differed in the row-sparing and arc-sparing groups, in that the expansion was preferentially into deprived, not spared areas. Single whisker stimulation in row-spared cases preferentially activated the corresponding barrel arc, while stimulation of one whisker in arc-spared cases produced elongated activation down the barrel row. Since whisker deflection normally has a net inhibitory effect on neighboring barrels, our data suggest that intracortical inhibition fails to develop normally in deprived cortical columns. Because thalamocortical projections are not affected by follicle ablation after P7, we suggest that the effects we observed are largely cortical, not thalamocortical.     

Structure of botulinum neurotoxin type D light chain at 1.65 A resolution: repercussions for VAMP-2 substrate specificity

The seven serotypes (A-G) of botulinum neurotoxins (BoNTs) function through their proteolytic cleavage of one of three proteins (SNAP-25, Syntaxin, and VAMP) that form the SNARE complex required for synaptic vesicle fusion. The different BoNTs have very specific protease recognition requirements, between 15 and 50 amino acids in length depending on the serotype. However, the structural details involved in substrate recognition remain largely unknown. Here is reported the 1.65 A resolution crystal structure of the catalytic domain of BoNT serotype D (BoNT/D-LC), providing insight into the protein-protein binding interaction and final proteolysis of VAMP-2. Structural analysis has identified a hydrophobic pocket potentially involved in substrate recognition of the P1' VAMP residue (Leu 60) and a second remote site for recognition of the V1 SNARE motif that is critical for activity. A structural comparison of BoNT/D-LC with BoNT/F-LC that also recognizes VAMP-2 one residue away from the BoNT/D-LC site provides additional molecular details about the unique serotype specific activities. In particular, BoNT/D prefers a hydrophobic interaction for the V1 motif of VAMP-2, while BoNT/F adopts a more hydrophilic strategy for recognition of the same V1 motif.     

Characterization of a 160 kD Photosystem II reaction center complex isolated from photoinhibited Dunaliella salina thylakoids

Photoinhibition in the green alga Dunaliella salina is accompanied by the formation of inactive Photosystem II reaction centers. In SDS-PAGE analysis, the latter appear as 160 kD complexes. These complexes are structurally stable, enough to withstand re-electrophoresis of excised gel slices from the 160 kD region. Western blot analyses with specific polyclonal antibodies raised against the D1 or D2 reaction center proteins provided evidence for the presence of both of these polypeptides in the re-electrophoresed 160 kD complex. Incubation of excised gel slices from the 160 kD region, under aerobic conditions at 4°C for a prolonged period of time, caused a break-up of the 160 kD complex into a 52 kD D1-containing and 80 and 26 kD D2-containing pieces. Western blot analysis with polyclonal antibodies raised against the apoproteins of CPI (reaction center proteins of PS I) did not show cross-reaction either with the 160 kD complex or with the 52, 80 and 26 kD pieces. The results show the presence of both D1 and D2 in the 160 kD complex and strengthen the notion of a higher molecular weight D1- and D2-containing complex that forms upon disassembly of photodamaged PS II units.Abbreviations Chl chlorophyll - PS II Photosystem II - D1 the 32 kD reaction center protein of PS II, encoded by the chloroplast psbA gene - D2 the 34 kD reaction center protein of PS II, encoded by the chloroplast psbD gene - CPI the 82 and 83 kD reaction center proteins of PS I, encoded by the chloroplast psaA and psaB genes - HL high light - LL low light This publication is dedicated to the memory of the late Professor Daniel Arnon, whom the first author will fondly remember for his many accounts of past scientific discovery and debate.